What is the recommended enzyme amount when using Taq DNA Polymerase or Green Taq DNA Polymerase?

0.5 U or more

How is Taq different from Green Taq?

The Green Taq contains normal Taq with special stabilizers. The Green Taq can be stable under RT for up to two weeks

Can Taq DNA Polymerase or Green Taq be used to amplify GC-rich amplicons?

No.

Can Taq DNA Polymerase be used in other buffers?

Taq DNA polymerase can have 100% activity in the supplied GenScript Taq Buffer. Additionally, the Taq DNA polymerase can also work well in other buffers. For your convenience, GenScript offers Taq buffer under the name 10x Taq Buffer (Cat. No. B0005)

Which buffer should I use if I want to control the level of magnesium (Mg2+) in the reaction? Does the presence of Mg2+ inhibit PCR?

This is a potential concern. For this reason we suggest to use Mg-free Taq DNA Polymerase (Cat. No. E00008).

How should I set up an amplification reaction using Taq DNA Polymerase?

add 5% DMSO, 1 M betaine, or both can be included in PCR reactions to improve results

Template degradation

Analyze the template on an agarose gel to check for possible degradation

PCR inhibitor contamination

Test the template with an established PCR system

Repeat template purification to remove the remainder alcohol, Phenol or other contamination

Cycle conditions

Change annealing temperature

Increase cycle number

Prolong the elongation time

Primer problem

Both primers must have the same concentration. The final concentration must be between 0.2 µM ~ 0.6 µM

Make sure that the primers are not degraded

Low enzyme concentration

Increase the amount of polymerase in 0.5 U steps

Low MgCl2 concentration

Increase the MgCl2 concentration in 0.25mM steps

Inadequate annealing temperature

Increase annealing temperature

Improper template concentration

Test serial dilution of template

Formation of primer dimers

When the temperature of the PCR cycle reaches 94°C insert PCR reaction tube and continue the program

PCR Cloning Kit

Answers:

What are the differences between the CloneEZ® kit and the PCR Cloning Kit?

CloneEZ® seamless cloning technology offers high efficiency, quick and direct cloning, bypassing all the tedious procedures of traditional cloning that often involve restriction, ligation, and sometimes phosphorylation. This technology works with any DNA sequence and any vector. The only requirement is that the PCR primers have a sequence containing 15 or more nucleotides homologous to the vector sequence.

Technical information is not available in the manual, why is that?

Currently, we are waiting for a patent on CloneEZ®. Once the patent is valid a more detailed explanation in the manual will be provided.

Why don't I generate sufficient colonies when the CloneEZ® reaction is transformed?

This may be caused by four factors.

The competent cells have low transformation efficiency.

Too much reaction mixture is used.

Presence of Inhibitory contaminants from PCR DNA or linearized vector.

The molar ratio of vector to insert is off.
Please refer to the manual for TROUBLESHOOTING.

Why do I generate incorrect colonies when the CloneEZ® reaction is transformed?

Two factors may be involved.

The cloning vector is not completely linearized.

The cloning reaction is contaminated with plasmids having the same antibiotic resistance.

What factors need to be considered when designing the primer?

Two sets of primers are used to amplify the gene of interest:

15-bp homology regions to the vector, flanking the cloning site into the insert.

Specific gene sequence. If the sequence has 5' overhang, the start of the 15 bp homology must begin from the 5' most extension to include the overhang; if the sequence has a 3' overhang, homology should begin where the DNA becomes double-stranded. Please refer to the manual for illustration of primer design.

What should the purity of my primer be to be compatible with the CloneEZ® cloning method?

Desalted oligos (from a qualified supplier) are suitable for cloning with the CloneEZ® PCR cloning Kit.

Can multiple fragments be cloned into a single vector using this kit?

Yes. CloneEZ® PCR Cloning Kit can be used for multiple fragment recombination if there are not many repeated sequences among multiple fragments. However, we recommend recombination one fragment at a time. Several performances of recombining 2 - 3 fragments demonstrated efficiency as low as 20%-30%, and lead to a direct repeat deletion.

Can I use CloneEZ® PCR Cloning Kit with my own vectors?

Yes. GenScript's CloneEZ® PCR Cloning Kit has been successfuly tested with several commercial and non-commercial vectors.

What factors need to be considered when using CloneEZ® PCR Cloning Kit with customers' vector?

We recommend linearizing your vectors before using CloneEZ® PCR Cloning Kit. Once linearized, the columned and gel-purified vector is ready for CloneEZ reaction.

Will the CloneEZ® reaction work more efficiently if I use primers that contain a longer than 15 base region of homology?

A 15-20 bp homology is recommended.

What is the stability of the enzyme included in CloneEZ® PCR Cloning Kit?

The liquid enzyme should be stored at -20°C for at least 12 months without activity loss.

Do not add more than 20 μl of reaction mixture to 50 μl of competent cells. Too much reaction mixture inhibits the transformation.

There are inhibitory contaminants from PCR DNA or from linearized vector. The molar ratio of vector to insert is off.

Both the PCR DNA and the linearized vector should be purified. Usually an insert/vector molar ratio of 2:1 is optimal. If the insert is as large as the linearized vector, a molar ratio of 1:1 can also be used.

Too long or short a recombination time

It is recommended to keep the recombination procedure within 30 min

The linearized cloning vector or primer is large.

Connect the product to the target step recombinant vector

The cloning vector is not completely linearized.

Gel-purify the linearized vector.

The cloning reaction is contaminated with plasmids having the same antibiotic resistance.

Purified PCR DNA may contain the template plasmid, so gel-purify the PCR DNA.

Most of the colonies contain no insert.

The cloning vector is not completely linearized.

Gel-purify the linearized vector.

The cloning reaction is contaminated with plasmids having the same antibiotic resistance.

Purified PCR DNA may contain the template plasmid, so gel-purify the PCR DNA.

Plasmid Purification

Answers:

Why is my plasmid DNA yield low?

Check if the bacteria was cultured properly,

Check if bacteria cells were resuspended completely.

Incubate the Elution Buffer at 30-60°C，this should help increase yields

Can the QuickClean II plasmid Miniprep Kit be used for isolating plasmid DNA from mammalian cells?

No. The Kit provides a fast, simple, and cost-effective plasmid miniprep method for 1–5ml of overnight cultures of E. Coli.

What is the recommended culture medium for the Plasmid miniprep kit?

LB culture medium

How do I know if my plasmid is a high- or low copy number type?

For example, a series of PUC vector is high-copy-number plasmid. You can know the different type of plasmid from their name.

How to resolve genomic DNA contamination in my plasmid prep?

If getting genomic DNA contamination in the prep, invert the tube gently after adding the Lysis Buffer.

Can I use QuickClean Miniprep kits for low-copy plasmids and cosmids?

The kit can purify the plasmid from low-copy plasmids, but from cosmids.

Are GenScript buffers with identical names in different kits the same?

Yes, GenScript buffers that have exactly the same name, are chemically identical and can be exchanged between kits. For example, wash solution and elution buffer from the QuickClean II PCR Purification Kit are exactly the same as wash solution and elution buffer from QuickClean II PCR Gel Extraction Kit.

The DNA eluted from the column is pure enough for most downstream applications. However, if the downstream applications are sensitive to salt carryover, it is better to wash the column twice prior to elution.

Can I use TE buffer or water to elute DNA from the column?

Elution Buffer is 2.5 mM Tris-HCl pH 8.5. TE buffer or water can also be used, but yield will be slightly lower. The pH of the elution solution is critical; buffers with a higher pH such as 8.5 or above will be more efficient to elute DNA from the column.

What additional consumables does the user need?

Ethanol is needed to prepare the wash solution for all three kits. Isopropanol is also needed for QuickClean II PCR or Gel Extraction Kit.

Can I extract and purify DNA from gels using TAE running buffer?

Yes. QuickClean II PCR or Gel Extraction Kit can be used to extract and purify DNA from gels using either TBE or TAE as the electrophoresis buffer.

Can I extract and purify DNA from low melting point (LMP) Agarose gels?

Yes. QuickClean II PCR or Gel Extraction Kit can be used to extract and purify DNA from low melting point (LMP) gels.

Problem

Solution

Obtain no plasmid DNA

If there is no plasmid DNA in the elution buffer, please check whether the ethanol had been added to wash buffer according to the volume marked on bottle label.

Low plasmid DNA yields

Please check if the bacteria was cultured properly

Please check if the bacteria cells were resuspended completely. 3. Incubate the Elution Buffer at 30～60°C，to help increase the yields.

Absorbance problem

Absorbance is the difference of the sample from the blank, please use the Elution Buffer to adjust the blank to a zero value and use it to dilute the sample.

If the ratio of OD260/ OD230 is too low, wash the spin column for one more time.

In the case of a low ratio of OD260-320/ OD280-320, there may be protein contamination. In this case please add Neutralization Buffer, and then centrifuge buffer with sufficient rotating speed, thus to make precipitation compact; be careful to pipette supernatant and avoid pipetting the precipitate.

If the ratio of OD260-320/ OD280-320 is too high, add more RNase A to Resuspension Buffer to a final concentration 100 μg/ml.

Electrophoresis problem

If there is genomic DNA in the result, invert the tube gently (step 3 and 4).

If there is RNA in the result, add more RNase A to Resuspension Buffer to a final concentration 100 μg/ml.